Asada Kiyoshi, Kaji Kosuke, Sato Shinya, Seki Kenichiro, Shimozato Naotaka, Kawaratani Hideto, Takaya Hiroaki, Sawada Yasuhiko, Nakanishi Keisuke, Furukawa Masanori, Kitade Mitsuteru, Moriya Kei, Namisaki Tadashi, Noguchi Ryuichi, Akahane Takemi, Yoshiji Hitoshi
Third Department of Internal Medicine, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Biology (Basel). 2020 Jun 3;9(6):117. doi: 10.3390/biology9060117.
Hepatic stellate cell (HSC) activation is essential for the development of liver fibrosis. Epigenetic machinery, such as DNA methylation, is largely involved in the regulation of gene expression during HSC activation. Although the pharmacological DNA demethylation of HSC using 5-aza-2'-deoxycytidine (5-aza-dC) yielded an antifibrotic effect, this drug has been reported to induce excessive cytotoxicity at a high dose. Hydralazine (HDZ), an antihypertensive agent, also exhibits non-nucleoside demethylating activity. However, the effect of HDZ on HSC activation remains unclear. In this study, we performed a combined treatment with 5-aza-dC and HDZ to obtain an enhanced antifibrotic effect with lower cytotoxicity.
HSC-T6 cells were used as a rat HSC cell line in this study. The cells were cultivated together with 1 µM 5-Aza-dC and/or 10 µg/mL of HDZ, which were refreshed every 24 h until the 96 h treatment ended. Cell proliferation was measured using the WST-1 assay. The mRNA expression levels of peptidylprolyl isomerase A (), an internal control gene, collagen type I alpha 1 (), RAS protein activator like 1 (), and phosphatase and tensin homolog deleted from chromosome 10 () were analyzed using quantitative reverse transcription polymerase chain reaction.
The percentage cell viability with 5-aza-dC, HDZ, and combined treatment vs. the vehicle-only control was 101.4 ± 2.5, 95.2 ± 5.7, and 79.2 ± 0.7 ( < 0.01 for 5-aza-dC and < 0.01 for HDZ), respectively, in the 48 h treatment, and 52.4 ± 5.6, 65.9 ± 3.4, and 29.9 ± 1.3 ( < 0.01 for 5-aza-dC and < 0.01 for HDZ), respectively, in the 96 h treatment. 5-Aza-dC and the combined treatment markedly decreased mRNA levels. Accordingly, the expression levels of and , which are antifibrotic genes, were increased by treatment after the 5-aza-dC and combined treatments. Moreover, single treatment with HDZ did not affect the expression levels of , , or . These results suggest that HDZ sensitizes to the antifibrotic effect of 5-aza-dC in HSC-T6 cells. The molecular mechanism underlying the sensitization to the antifibrotic effect of 5-aza-dC by HDZ remains to be elucidated. The expression levels of rat equilibrative nucleoside transporter genes (, , and ) were not affected by HDZ in this study.
Further confirmation using primary HSCs and in vivo animal models is desirable, but combined treatment with 5-aza-dC and HDZ may be an effective therapy for liver fibrosis without severe adverse effects.
肝星状细胞（HSC）激活是肝纤维化发展的关键环节。表观遗传机制，如DNA甲基化，在很大程度上参与了HSC激活过程中的基因表达调控。虽然使用5-氮杂-2'-脱氧胞苷（5-aza-dC）对HSC进行药理学DNA去甲基化产生了抗纤维化作用，但据报道该药物在高剂量时会诱导过度的细胞毒性。肼屈嗪（HDZ）是一种抗高血压药物，也具有非核苷去甲基化活性。然而，HDZ对HSC激活的影响尚不清楚。在本研究中，我们进行了5-aza-dC和HDZ的联合治疗，以获得增强的抗纤维化效果且降低细胞毒性。
本研究使用HSC-T6细胞作为大鼠HSC细胞系。将细胞与1 μM 5-氮杂-dC和/或10 μg/mL HDZ一起培养，每24小时更换一次，直至96小时治疗结束。使用WST-1法测量细胞增殖。使用定量逆转录聚合酶链反应分析肽基脯氨酰异构酶A（一种内参基因）、I型胶原α1、RAS蛋白激活样1和10号染色体缺失的磷酸酶及张力蛋白同源物的mRNA表达水平。
在48小时治疗中（5-aza-dC、HDZ和联合治疗组与仅用溶剂对照组相比）细胞活力百分比分别为101.4±2.5、95.2±5.7和79.2±0.7（5-aza-dC组P<0.01，HDZ组P<0.01）；在96小时治疗中分别为52.4±5.6、65.9±3.4和29.9±1.3（5-aza-dC组P<0.01，HDZ组P<0.01）。5-aza-dC和联合治疗显著降低了mRNA水平。因此，5-aza-dC和联合治疗后，抗纤维化基因和的表达水平通过治疗而升高。此外，单独使用HDZ治疗不影响、或的表达水平。这些结果表明HDZ使HSC-T6细胞对5-aza-dC的抗纤维化作用敏感。HDZ使细胞对5-aza-dC抗纤维化作用敏感的分子机制仍有待阐明。在本研究中，大鼠平衡核苷转运体基因（、和）的表达水平不受HDZ影响。
虽然需要使用原代HSC和体内动物模型进行进一步证实，但5-aza-dC和HDZ联合治疗可能是一种治疗肝纤维化且无严重不良反应的有效疗法。
